Synthesis Of Firststrand cDNAs From Cell Or Tissue Type A Or B

Protocol A. Synthesis of First-Strand cDNAs from mRNAs

It is recommended that two reactions that will be carried out separately all the way to the end be set up in two microcentrifuge tubes. The benefit is that if one reaction is stopped by an accident during the experiments, the other reaction can be used as a back-up. Otherwise, it is necessary to start over again, thus wasting time and money. A standard reaction is total 25 ml using 0.2 to 2 mg mRNA or 5 to 10 mg total RNAs. For each additional 1 mg mRNA, increase the reaction volume by 10 ml. A control reaction is recommended to be carried out whenever possible under the same conditions.

1. Anneal 2 mg of mRNAs as templates with 1 mg of primer of oligo(dT), or with 0.7 mg of primer-adapter of oligo(dT)-Not I/Xba I in a 1.5-ml sterile, RNase-free microcentrifuge tube. Add nuclease-free dd.H2O to 15 ml. Heat the tube at 70°C for 5 min to denature the secondary structures of mRNAs and allow it to slowly cool to room temperature to complete the annealing. Briefly spin down the mixture.

2. To the annealed primer-template, add the following in the order listed. To prevent precipitation of sodium pyrophosphate when adding it to the reaction, the buffer should be preheated at 40 to 42°C for 4 min prior to the addition of sodium pyrophosphate and AMV reverse transcriptase. Gently mix well following each addition.

First-strand 5X buffer, 5 ml rRNasin ribonuclease inhibitor, 50 units (25 units/mg mRNA) 40 mM sodium pyrophosphate, 2.5 ml AMV reverse transcriptase, 30 units (15 units/mg mRNA) Add nuclease-free dd.H2O to a final 25 ml

Function of components: The 5X reaction buffer contains components for the synthesis of cDNAs. Ribonuclease inhibitor serves to inhibit RNase activity and to protect the mRNA templates. Sodium pyrophosphate suppresses the formation of hairpins that are commonly generated in traditional cloning methods, thus avoiding the S1 digestion step that is difficult to carry out. AMV reverse transcriptase catalyzes the synthesis of the firststrand cDNAs from mRNA templates according to the rule of complementary base pairing.

3. Transfer 5 ml of the mixture to a fresh tube, as a tracer reaction, which contains 1 ml of 4 mCi of [a-32P]dCTP (>400 Ci/mmol, less than 1 week old). The synthesis of the first-strand cDNAs will be measured by trichlo-roacetic acid (TCA) precipitation and alkaline agarose gel electrophoresis using the tracer reaction.

Caution: [a-32P]dCTP is a toxic isotope. Gloves should be worn when carrying out the tracer reaction, TCA assay and gel electrophoresis. Waste materials such as contaminated gloves, pipette tips, solutions and filter papers should be put in special containers for disposal of waste radioactive materials.

4. Incubate both reactions at 42°C for 1 to 1.5 h, and then place the tubes on ice. At this point, the synthesis of the first-strand cDNAs has been completed. To the tracer reaction mixture, add 50 mM EDTA solution up to a total volume of 100 ml and store on ice until used for TCA incorporation assays and analysis by alkaline agarose gel electrophoresis.

5. To remove RNAs completely, add RNase A (DNase-free) to a final concentration of 20 mg/ml or 1 ml of RNase (Boehringer Mannheim, 500 mg/ml) per 10 ml of reaction mixture. Incubate at 37°C for 30 min.

6. Extract the cDNAs from the unlabeled reaction mixture with one volume of TE-saturated phenol/chloroform. Mix well and centrifuge at 11,000 x g for 5 min at room temperature.

7. Carefully transfer the top, aqueous phase to a fresh tube. Do not take any white materials at the interphase between the two phases. To the supernatant, add 0.5 volume of 7.5 M ammonium acetate or 0.15 volume of 3 M sodium acetate (pH 5.2), mix well and add 2.5 volumes of chilled (at -20°C) 100% ethanol. Gently mix and precipitate cDNAs at -20°C for 2 h.

8. Centrifuge in a microcentrifuge at 12,000 x g for 5 min. Carefully remove the supernatant, briefly rinse the pellet with 1 ml of cold 70% ethanol. Centrifuge for 4 min and carefully aspirate the ethanol.

9. Dry the cDNAs under vacuum for 15 min. Dissolve the cDNA pellet in 15 to 25 ml of TE buffer. Take 2 ml of the sample to measure the concentration of cDNAs prior to the next reaction. Store the sample at -20°C until use.

Protocol B. Trichloroacetic Acid (TCA) Assay and Calculation of cDNA-Yield

1. Spot 4 ml of the first-strand cDNA tracer reaction mixture on glass fiber filters and air-dry. These will be used to obtain the total counts per minute (cpm) in the sample.

2. Add another 4 ml of the same reaction mixture to a tube containing 100 ml of carrier DNA solution (1 mg/ml) and mix well. Add 0.5 ml of 5% TCA and mix by vortexing and precipitate the DNA on ice for 30 min.

3. Filter the precipitated cDNA through glass fiber filters and wash the filters four times with 6 ml cold 5% TCA. Rinse the filters once with 6 ml of acetone or ethanol and air dry. This sample represents incorporated cpm in the reactions.

4. Place the filters at step 1 and step 3 in individual vials and add 10 to 15 ml of scintillation fluid to cover each filter. Count both total and incorporated cpm samples according to the instructions of an appropriate scintillation counter. The cpm can also be counted by Cerenkov radiation (without scintillant).

5. Calculate the yield of first-strand cDNA as follows:

Percentage incorporated = incorporated cpm/total cpm x 100 dNTP incorporated (nmol) = 4 nmol dNTP/ml x reaction vol.(ml) x % incorporation/100 cDNA synthesized (ng) = nmol dNTP incorporated x 330 ng/nmol % mRNA converted to cDNA = ng cDNA synthesized/ng mRNA in reaction.

Protocol C. Analysis of cDNAs by Alkaline Agarose Gel Electrophoresis

The quality and the size range of the synthesized cDNAs in the first-strand cDNA reactions should be checked by 1.4% alkaline agarose gel electrophoresis using the tracer reaction samples.

1. Label iHind III fragments with 32P in a fill-in reaction that will be used as DNA markers to estimate the sizes of cDNAs. Add the following components to a sterile Eppendorf tube in the order shown:

Hind III 10X buffer, 2 ml dGTP, 0.2 mM

[a-32P]dCTP, 2 mCi iHind III markers, 1 mg

Klenow DNA polymerase, 1 unit

Add dd.H2O to a final volume of 20 ml.

2. Mix well after each addition and incubate the reaction for 15 min at room temperature. Add 2 ml of 0.2 M EDTA to stop the reaction. Transfer 6 ml of the sample directly to 6 ml of 2X alkaline buffer and store the remainder at -20°C.

3. Dissolve 1.4% (w/v) agarose in 50 mM NaCl, 1 mM EDTA solution and melt it in a microwave for a few minutes. Allow the gel mixture to cool to 50°C and pour the gel into a mini-electrophoresis apparatus. Allow the gel to harden and equilibrate it in alkaline gel running buffer for 1 h before electrophoresis.

4. Transfer the same amount of each sample (50,000 cpm) to separate tubes and add an equal volume of TE buffer to each tube. Add one volume of 2X alkaline buffer to each tube.

5. Carefully load the samples into appropriate wells and immediately run the gel at 7 V/cm until the dye has migrated to about 2 cm from the end.

6. When electrophoresis is complete, soak the gel in five volumes of 7% TCA at room temperature until the dye changes from blue to yellow. Dry the gel on a piece of Whatman 3MM paper in a gel dryer or under a weighted stack of paper towels for 6 h. The 7% TCA is used here to neutralize the denatured gel.

7. Wrap the gel with a piece of SaranWrap™ and expose to x-ray film at room temperature or at -70°C with an intensifying screen for 1 to 4 h.

Note: A successful synthesis of cDNAs should have a signal range from 0.15 to 10 kb with a sharp size range of 1.5 to 4 kb (Figure 3.4).

Materials Needed

50 mM EDTA

7.5 M Ammonium acetate

Chloroform:isoamyl alcohol (24:1)

2 M NaCl

0.2 M EDTA

1 mg/ml Carrier DNA (e.g., salmon sperm) Trichloroacetic acid (5% and 7%)

FIGURE 3.4 Analysis of first-strand cDNAs using agarose electrophoresis.

First-Strand 5X Buffer

250 mM KCl

2.5 mM Spermidine

50 mM MgCl2

50 mM DTT

5 mM Each of dATP, dCTP, dGTP and dTTP

TE Buffer

TE-Saturated Phenol/Chloroform

Thaw phenol crystals at 65°C and mix an equal part of phenol and TE buffer. Mix well and allow the phases to separate at room temperature for 30 min. Take 1 part of the lower, phenol phase to a clean beaker or bottle and mix with 1 part of chloroform:isoamyl alcohol (24:1). Mix well and allow phases to be separated, and store at 4°C until use.

Alkaline Gel Running Buffer (Fresh) 30 mM NaOH 1 mM EDTA

2X Alkaline Buffer 20 mM NaOH 20% Glycerol

0.025% Bromophenol blue (use fresh each time)

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